Agents, which inhibit apoptosis in cells that are involved in wound healing

ABSTRACT

The invention relates to the use of substances as a fundamental constituent in wound healing agents. The invention is characterised in that said substances bond to either IAP and/or integrin α v β 3  and/or thrombuspondin-1 in such a way that the bond between thrumbospondin-1 and IAP and/or integrin α v β 3  is inhibited.

This is the U.S. national phase of International Application No.PCT/EP02/01828 filed Feb. 21, 2002, the entire disclosure of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to agents which have an anti-apoptoticeffect on cells involved in wound healing. In particular, the presentinvention relates to the use of substances which bind either to IAPand/or to integrin α_(v)β₃ and/or to thrombospondin 1 in such a way thatthe binding between thrombospondin 1 and IAP and/or integrin α_(v)β₃ isinhibited, thus reducing the rate of apoptosis of cells associated withwound healing, for producing medicaments which are suitable for thetreatment of traumatic conditions, preferably chronic wounds. Preferredexemplary embodiments encompass the amino acid sequences depicted in SEQID Nos 1-11, and peptidomimetics derived therefrom. The presentinvention further relates to cells having a significantly reduced rateof apoptosis, it being possible to use these cells preferably asconstituent of a so-called artificial skin which has already beendescribed in the prior art, and the skin being mechanically tensionedbefore application of this skin to the wound, and the correspondingcells preferably being treated additionally with the agents of theinvention.

2. Description of Related Technology

The process of wound healing consists of three phases during which theinjured tissue is repaired and is thus regenerated, after which the newtissue becomes manifest as scar.

These three phases are classified in the following way:

-   (a) inflammatory phase which starts after 0 to 3 days-   (b) a subsequent cellular proliferation phase of 3 to 12 days, and-   (c) a restorative phase of 3 days to about 6 months.

In the inflammatory phase, inflammatory cells, mainly neutrophils,collect at the wound site, followed by lymphocytes, monocytes and evenlater macrophages.

During the proliferation phase, the so-called granulation tissue (highlyvascularized connective tissue which forms during the healing of wounds,ulcers inter alia) is formed in the injured area. An important part isplayed in this by, in particular, fibroblasts and epithelial cells(re-epithelization), besides other cell types. The fibroblasts producethe collagen which is important for wound healing.

Ascorbic acid (vitamin C) is essential for the formation of collagen. Ithas already been shown in several investigations that the use ofascorbic acid activates the reduced proliferative activity and thecollagen synthetic activity of aged skin fibroblasts, and thereforewound healing is improved. However, the mode of action has not beenshown, but inter alia an involvement of vitamin C in lipid oxidationprocesses is described.

During the re-epithelization, the epithelial cells proliferate andmigrate from the edges of the wound into the tissue. It has beenpossible to show in this connection that re-epithelization can bepromoted by wound dressings which represent a moisture barrier.

The concluding phase of wound healing is characterized by thereplacement of the granulation tissue by collagen and elastin fibers andthe devascularization of the granulation tissue (i.e. formation of scartissue). Recent studies have moreover shown that topical application ofantioxidants such as alpha-tocopherol reduces scar formation andnormalizes blood coagulation during therapy.

Fibroblasts and epithelial cells in particular therefore play animportant part in wound healing. The low proliferation activity of thefibroblasts and the small number thereof impedes favorable woundhealing. In many cases there is use, besides antiinflammatorysubstances, also of substances such as growth factors which are intendedto enhance the proliferation abilities or the synthetic activity of thecells involved.

Apoptosis (synonym: programmed cell death) is an irreversible process.An apoptotic cell inevitably dies. Apoptotic fibroblasts have beensuggested to have a negative role in wound healing, but no way has yetbeen proposed for having a beneficial influence on the rate of apoptosisof fibroblasts during wound healing by bringing particular medicaments,i.e. molecular remedies, into contact with the fibroblasts.

European patent application EP-A-0 903 149 describes a method foridentifying apoptosis-inducing substances in immune cells. It was shownthat substances which bind to the integrin-associated protein (IAP or CD47) on the surface of immune cells may have the ability to induceapoptosis. The mechanisms of action were not described.

It has already been proposed that IAP is involved in the formation of aspecific calcium channel (Schwartz, M. A. et al., Journal of BiologicalChemistry, 268:27, 19931-19934). No mention was made of a role of thishypothetical calcium channel in the induction of apoptosis.

The applicant's international patent application WO 01/33218 A1 ofearlier priority, the full contents of which are incorporated in thepresent application by reference, describes a method for identifyingapoptosis-inhibiting substances and the use of such substances forproducing medicaments for the treatment of vascular disorders and asactive principle in a pharmaceutical preparation for the treatment ofvascular disorders. A use of such substances for producing medicamentsfor promoting wound healing or as active principle of a pharmaceuticalpreparation for the treatment of wounds was not described.

One of the most modern approaches to the treatment of chronic wounds isthe use of a so-called living skin as skin substitute or wound-coveringmeans. There has already been a large number of patent applications inthis area. Mention may be made here representatively to EP 1 005 873 ofIsotis N.V. or the international patent application WO 99/43787 ofAdvanced Tissue Sciences Inc. The principle of the production of such anartificial skin is to grow either autologous or heterologous cells (skincells) on a biocompatible membrane and then use this artificial skin asremedy. However, it has not previously been described that it isbeneficial or necessary to inhibit the apoptosis of the cells used inorder to ensure a more favorable progress of the healing. Neitherstretching of the artificial skin nor the addition of anti-apoptoticagents is proposed. A negative effect of apoptosis on the progress ofhealing is not discussed in these patent applications and is not madeobvious to the skilled worker.

A healing influence of chronic traumatic conditions or large-area and/ordeep wounds, like those frequently occurring in burn injuries, inparticular is possible at present, despite the prior art mentioned, onlywith difficulty.

There is thus still a great need in the art for improved means whichbeneficially influence wound healing, and in particular also the healingof chronic wounds or large-area wounds or burn injuries of the skin.

DETAILED DESCRIPTION

The present invention is therefore based on the object of providingsubstances and/or agents which are suitable for producing a medicamentor a means for the treatment of traumatic conditions and/or burninjuries.

This is achieved, as are other not explicitly mentioned objects which,however, can be deduced or inferred directly from the relationshipsdiscussed in the introduction, according to the invention. Expedientmodifications of the use according to the invention are described.

The object is achieved in particular by the use of substances which bindeither to IAP and/or to integrin α_(v)β₃ and/or to thrombospondin 1 insuch a way that the binding between thrombospondin 1 and IAP and/orintegrin α_(v)β₃ is inhibited, and which reduce the rate of apoptosis ofcells associated with wound healing, for producing medicaments whichcomprise as active ingredient at least one such substance, and which areemployable for the treatment of wounds, in particular for the treatmentof chronic wounds and/or burn injuries.

A medicament for the treatment of said wound injuries can be produced byinitially carrying out an identification cation method of stages (i) to(v), wherein:

-   (i) cells which express both IAP and the integrin α_(v)β₃ are    cultured,-   (ii) the cells are caused to produce an apoptosis-inducing    substance, and/or a substance or substances inducing apoptosis    is/are added,-   (iii) a test substance as claimed in any of claims 5 to 9 added,-   (iv) the rate of apoptosis is measured, and-   (v) test substances which bring about a reduced rate of apotosis are    selected, and then-   (vi) the test substances identified in this way (identificates) are    mixed with a pharmaceutically suitable carrier.

The substances (identificates) which can be used according to theinvention particularly preferably inhibit the apoptosis of fibroblastsand/or epithelial cells.

The present invention additionally preferably encompasses medicamentswhich comprise as active ingredient one such substance and one or more,optionally different fibroblast growth factors, such as, for example,basic fibroblast growth factor (bFGF), and which are employable for thetreatment of wounds, and in particular for the treatment of chronicwounds and/or burn injuries.

The invention additionally encompasses preferably the use of thesesubstances for the treatment of traumatic conditions, in particular forthe treatment of chronic wounds.

The object of the invention is achieved in particular by the provisionof substances which comprise peptides having one of the amino acidsequences depicted in SEQ ID Nos 1 to 11.

A preferred exemplary embodiment of the present invention comprises inthis connection apoptosis-inhibiting substances which include amino acidsequences of the general formula (1):R-A1-Y-V-V-M,where A1 is A, D, E, G, M, N, T, W or Y, or pharmaceutically acceptablesalts of these substances.

For the purposes of the present invention, the internationally customaryone-letter code for amino acids is used, and thus A is alanine (Ala), Cis cysteine (Cys), D is aspartic acid (Asp), E is glutamic acid (Glu), Fis phenylalanine (Phe), G is glycine (Gly), L is leucine (Leu), M ismethionine (Met), N is asparagine (Asn), P is proline (Pro), R isarginine (Arg), S is serine (Ser), T is threonine (Thr), V is valine(Val), W is tryptophan (Trp) and Y is tyrosine (Tyr).

In a particularly preferred aspect, the present invention thereforerelates to apoptosis inhibiting substances, preferably proteins orpeptides, which comprise one of the peptide sequences shown in SEQ IDNo. 1 to SEQ ID No. 11, or the corresponding pharmaceutically acceptablesalts thereof.

The present invention further relates to the use of the substances ofthe invention, preferably proteins or peptides comprising at least oneof the amino acid sequence depicted in SEQ ID No. 1 to SEQ ID No. 11 forproducing medicaments, in particular for producing medicaments for thetreatment of traumatic conditions, and very particularly preferably inthis connection chronic wounds or severe burn injuries.

Surprisingly, the inventors have shown that peptides comprising an aminoacid sequence represented by formula (1) inhibit apoptosis to anextremely great extent. These peptides are outstandingly suitable inparticular for inhibiting TSP-1 induced apoptosis.

The following peptides/peptide sequences are provided as veryparticularly preferred exemplary embodiments of the present invention:

1. R-A-Y-V-V-M (SEQ ID No. 1) 2. R-W-Y-V-V-M (SEQ ID No. 2) 3.R-Y-Y-V-V-M (SEQ ID No. 3) 4. R-E-Y-V-V-M (SEQ ID No. 4) 5.K-R-A-Y-V-V-M-W-K-K (SEQ ID No. 5) 6. K-R-E-Y-V-V-M-W-K-K (SEQ ID No. 6)7. R-G-Y-V-V-M (SEQ ID No. 7) 8. R-M-Y-V-V-M (SEQ ID No. 8) 9.R-T-Y-V-V-M (SEQ ID No. 9) 10. R-N-Y-V-V-M (SEQ ID No. 10) 11.R-D-Y-V-V-M (SEQ ID No. 11)

In a further preferred embodiment of the present invention, cellsinvolved in wound healing, preferably fibroblasts or epithelial cells,are cultivated as a so-called artificial skin already known in the art,and the latter is used according to the invention as wound-healingmeans, in which case this artificial skin is, after growth of the cells,mechanically tensioned and/or in which case the corresponding cells aretreated, before or after application of the artificial skin to the siteof the wound, with the agents of the invention, and where the proportionof apoptotic cells in the total number of cells can be reduced by atleast 10%, preferably by at least 50%, particularly preferably by atleast 75% and very particularly preferably by at least 90%, comparedwith an artificial skin which has not been mechanically tensioned andhas not been treated with the agents of the invention.

This entails this skin being stretched, preferably after growth of thecells on the biocompatible membrane or immediately before application ofthe skin to the wound, by 10-90%, and application of the agent of theinvention preferably 8-24 h after the stretching.

Surprisingly, the aforementioned inventors have shown that the bindingof thrombospondin-1 (TSP-1) to IAP and/or the integrin α_(v)β₃ inducesapoptosis in fibroblasts (examples 6 and 7). The correspondinginvestigations were carried out on MRC-5 fibroblasts (ATCC No.:CCL-171). The rate of apoptosis is determined as shown in example 3.

It was additionally possible, surprisingly, to show that the TSP-1 isproduced by the fibroblasts themselves, and thus the apoptosis isself-induced or spontaneous (example 8 and 9). These investigations werecarried out in conventional static cell cultures. The latter aredistinguished by the absence of flows in the cell culture medium.However, it was possible unexpectedly to show that TSP-1 is not producedby fibroblasts in a dynamic cell culture (cultivation in the cone andplate shear apparatus, see example 1), i.e. under conditions with whichthe cells are confronted by a flowing cell culture medium, and apoptosisoccurs to only a very small extent or not at all in this cell culture(example 5 and 8). This was highly surprising also because it cannot beassumed that fibroblasts are exposed under natural conditions to shearstresses caused by liquid flows.

Supplementation of fresh medium with TSP-1 causes an increase inspontaneous apoptosis in statically cultivated fibroblasts (example 6).This increase corresponds approximately to the effect of staticallyconditioned medium (example 5). The term conditioned medium means here acell culture medium which has previously already been used to cultivateother cells. This medium is distinguished by the fact that solublemediators, e.g. growth factors, hormones, etc., which are produced bycells during their cultivation, are dissolved therein. This result showsthat statically conditioned medium has the ability to induce apoptosisvia a mediator such as TSP-1. The production of conditioned medium isexplained in example 2.

It was possible to show by the use of an anti-TSP-1 antibody which bindsto and thus neutralizes TSP-1 that TSP-1 is the mediator of theapoptosis of fibroblasts (example 6). The effect of added TSP-1 can besuppressed, just like the effect of statically conditioned medium, byaddition of a polyclonal antiserum against TSP-1 and by addition of amonoclonal anti-TSP-1 antibody (example 6).

The TSP-1 secretion rates were determined for dynamic and staticpost-confluent cultures (example 8 and 9).

The term “static cell culture (conditions)” means here a cultivation ofcells under conditions with which invariable, i.e. consistentlydirected, laminar flows do not occur in the cell culture mediumsurrounding the cells. The “static cell culture conditions” in the senseused herein thus include cell culture conditions under which turbulentor variable laminar flows, that is to say, for example, those withchanging directions of flow or even with reversal of flow, occur. Theterm “dynamic cell culture (conditions)” means here cell cultureconditions with which only consistently directed, laminar flowconditions prevail in the cell culture medium, i.e. cell cultureconditions like those which can be achieved in the prior art for examplewith the aid of cultivation in a cone and plate shear apparatus (seeexample 1). It is clear that in the in vivo situation liquid flows donot occur with fibroblasts, because the connective tissue, in which thefibroblasts are chiefly located, is not a liquid-filled compartment.

The shear stresses acting on the cultivated cells in the cell culturevary with the flow conditions. A consistently directed laminar flowresults in a shear stress which is greater than 0.001 dyn/cm² and whosevector sum is greater than under variable flow conditions with changingdirections of flow. Static cell culture (conditions) are distinguishedby distinctly smaller (<0.001 dyn/cm²) or absolutely no stress. Dynamiccell cultures show shear stresses of >0.001 dyn/cm² or a Reynolds numberof >0.1. Turbulent flows may occur at Reynolds numbers of >200 (−1 000)(depending on the geometry of the flow chamber) and, like static orvariable flow conditions, no longer have a protective character inrelation to the induction of apoptosis. Various cultivation methods wereused for investigations which led to the present invention. These aredescribed in example 1.

Addition of TSP-1 to a dynamic fibroblast cell culture surprisingly hasno effect on apoptosis, in contrast to static culture (example 5). Thisshows that apoptosis depends not only on the production of TSP-1 but, onthe contrary, also on the occurrence or accessibility of specificreceptors on the surface of the cells. It was surprisingly possible toshow in this connection that the integrin α_(v)β₃ receptor is detectableon statically and dynamically cultivated cells, whereas the IAP receptoris expressed in detectable quantities only in static culture.

It is known that TSP-1 binds to the integrin α_(v)β₃. The binding ofTSP-1 to the α_(v)β₃ integrin is mediated by an RGD sequence motif. Acyclic RGD peptide which binds to the α_(v)β₃ integrin is marketed byBachem Biochemica GmbH, Heidelberg, Germany.

A further possible interaction of TSP-1 with a receptor on fibroblastsis binding to IAP via the C-terminal cell-binding domain (CBD). TheC-terminal cell-binding domain (CBD) is a domain of TSP-1 whichinteracts specifically with IAP. A truncated TSP-1 which consists onlyof this C-terminal cell-binding domain and binds to IAP is marketed byBachem Biochemica GmbH, Heidelberg, Germany, as CBD peptide.

Addition of the CBD peptide and of the cyclic RGD peptide surprisinglyled to a marked increase in the rate of apoptosis, which was at asimilar level to the increase in the rate of apoptosis by addition ofTSP-1 (example 7). It follows from this that only combined giving ofboth peptides to IAP and the integrin α_(v)β₃ is effective forapoptosis.

It was thus possible to show, surprisingly, that the activity of TSP-1for inducing spontaneous apoptosis is mediated exclusively via thebinding to IAP and α_(v)β₃ (example 10).

Since fibroblasts are not normally exposed to liquid flows in the invivo situation, the inventors named previously have further investigatedwhether other mechanical forces are also able to inhibit theself-induced apoptosis of fibroblasts.

Corresponding investigations were carried out on fibro-blasts culturedon collagen gels which had been isometrically contracted with ascorbicacid (example 11). It was surprisingly found in this case thatcultivation under tension inhibits spontaneous apoptosis. It wasadditionally found, surprisingly, that relaxation of such fibroblastscultivated with application of mechanical tension leads to a very markedincrease in the rate of apoptosis of the fibroblasts (example 12). Theresults achieved with addition of thrombospondin-1, anti-TSP1 andanti-receptor antibodies support these findings and clearly show that,surprisingly, the application of mechanical tension inhibits apoptosis,exactly like dynamic cultivation. The corresponding results are shown inexample 13.

In order to preclude an effect of the collagen used and of the ascorbicacid used, all the corresponding investigations were also carried outwith cells cultivated on contractile silicone sheets (cultivation in thestretching apparatus, examples 16and 17). It was additionally possibleto preserve the mechanical tension by freezing the actin fibers of thecytoskeleton in previously tensioned cells with chondramide A (example14) (chondramide A is an inhibitor of the depolymerization of F-actin).Expression of the proteins IAP and α_(v)β₃ as a function of mechanicalforce (examples 15and 18) was investigated in immunochemicalinvestigations. It was shown that the expression of IAP correlates withthe induction of apoptosis.

The aforementioned inventors were able by the investigations describedabove to show for the first time here that mechanical forces on theirown are sufficient for regulating apoptosis. The underlying molecularmechanisms were discovered for the first time by the aforementionedinventors.

Surprisingly, and in an entirely unpredictable manner, it is thereforepossible to employ substances usable according to the invention in orderto ensure a particularly good result of wound healing.

In addition, substances usable according to the invention and mechanicalforces can be employed in combination in order to ensure a particularlygood result of wound healing.

This is because the presented results show that the actual cause of theinduction of apoptosis is the absence of mechanical forces acting on thecells, the nature of these forces being immaterial. This causes thefibroblasts to secrete TSP-1, subsequently leading to the induction ofapoptosis through the interaction with the integrin α_(v)β₃ and IAP. Thedecrease in the fibroblast population (and other cells) leads to theprogress of healing being retarded or absent.

For the purposes of the present invention, the apoptosis rate means theproportion of apoptotic cells in the total number of cells. A substanceusable according to the invention is regarded as having anti-apoptoticactivity if it is possible by using it in the cell culture to reduce therate of apoptosis, compared with a cultivation carried out underidentical conditions but without addition of precisely this substance,by 10%, preferably by at least 50%, particularly by at least 75% andvery particularly preferably by at least 90%.

Such substances can be identified by:

-   (i) culturing cells which express both IAP and the integrin α_(v)β₃,-   (ii) causing the cells to produce an apoptosis-inducing substance,    and/or adding a substance or substances inducing apoptosis,-   (iii) adding the test substance,-   (iv) measuring the rate of apoptosis, and-   (v) selecting those substances whose addition to the cell culture    results in a reduced rate of apoptosis,

To carry out the corresponding experiments, the cells are cultivated inthe suitable culture media in suitable cell culture vessels. These arenormally standard media generally known in the prior art. For example,the cells are cultivated in DMEM, M-199, IF basal media etc. Suitableculture media are now available for virtually all cells and cell lines.Growth factors and hormones such as, for example, fetal calf serum (FCS)can be added to the culture medium before starting the culture.Mammalian cells are usually cultivated at 37° C. in a 5% CO₂ atmospherewhich, in connection with the buffers used in the cell culture media,e.g. sodium carbonate buffers, makes it possible to stabilize the pH ofthe cell culture medium. A further possibility is to add to the cellculture media, before starting the cultivation, antibiotics whichinteract specifically with prokaryotic contaminating microorganisms andinhibit their growth, but leave the growth of the eukaryotic cellsvirtually unaffected and thus protect the cell culture fromcontamination. Further hints and information for cell culturing can befound in standard works, e.g. Zell- und Gewebekultur, 3rd edition, ToniLindl, Jörg Bauer, (1994), Gustav Fischer Verlag.

Suitable culture conditions for cultivating fibroblasts are indicated inexample 1.

Apoptosis mediators can be added to the cell culture for induction orincreasing the rate of apoptosis.

This is because the spontaneous rate of apoptosis in a static cellculture is relatively low compared with rates of apoptosis induced bymediators such as TNF-α. The rates of apoptosis reached at thespontaneous rate of apoptosis induced by the static cell culturecondition are in the range from 0.5% to 12% of the total cells. In thecase of apoptosis induced by mediators, rates of apoptosis of up to 100%may be reached.

These mediators are hormones and other substances having apoptoticactivity. These mediators are added in dissolved form to the cellculture, and it should be noted that the solutions used must be sterile.The sterility of the solutions can be achieved in various ways,preferably by heat treatment (autoclaving at 2 bar and 120° C.) or, ifthis method is unsuitable because of a particular sensitivity of themediator to heat, by sterilizing filtration, for example using Nalgenedisposable sterilizing filters. Methods of this type for sterilizingadditions to cell culture are well known to the skilled worker.

The added test substances are preferably monoclonal antibodies, antibodyfragments, polyclonal antibodies and peptides. Further preferredsubstances which can be investigated as test substances in the testsystem of the invention are low molecular weight compounds. Suchcompounds often have only slight or no side effects if they are employedas active principle in a pharmaceutical composition. A further advantageof such substances is the possibility of oral administration. However,it is also possible to add other substances suspected of being able todisplay an anti-apoptotic effect. These substances are preferablyadministered in dissolved form. The solvents in this case must becompatible with the cell culture. These test substances are thereforepreferably dissolved in buffer solutions which have generally becomewidely used in cell culture. Examples thereof are phosphate buffers,sodium carbonate buffers and others. The dissolved test substances arepreferably sterilized by filtration (sterilized) by sterilizingfiltration (Nalgene disposable sterilizing filters) before addition tothe cell culture in order to remove contaminating microorganisms orspores of fungi and undissolved constituents.

The dissolved test substance is preferably equilibrated, i.e. adjustedto the temperature of the cell culture, before addition to the cellculture. The volumes depend on the concentration which is to be reachedof the test substance employed, and the volumes are preferably small sothat no dilution effects occur in the cell culture media. The methodswhich can be used to introduce such test substances into cell cultures,preferably in the dissolved state, are well known to the skilled worker.

The decrease in the rate of apoptosis can be determined by any suitablemeasurement method. The methods described in DE 199 52 960.4 canpreferably be employed for the present purposes. A particularly suitableearly indicator is measurement of the nonappearance of the calciuminflux into the cell through use of intra-cellular calcium indicators(see example 4). A method suitable for determining the exact rates ofapoptosis is staining of the DNA of apoptotic cells and subsequentmorphometric cell nucleus analysis or analysis of the cellular DNAcontent in a flow cytometer. An example of a suitable fluorescent dye isDAPI. These methods are well known in the art and are generally used fordetecting apoptotic cells. If the number of apoptotic cells decreasesafter the use of a potential inhibitor of apoptosis by more than 10%,preferably more than 50%, particularly preferably by more than 75%, andvery particularly preferably by more than 90%, based on the number ofapoptotic cells in the test system in static culture, where appropriateafter addition of an apoptosis-inducing substance, then this substanceis regarded according to the invention as an inhibitor of apoptosis inthe cells used. The non-appearance of the calcium influx into the cellcan be used as an early indicator for screening purposes. Substanceswhich lead to nonappearance of the calcium influx must subsequently bechecked for their effect on apoptosis inhibition by DAPI staining (seeexample 4).

It is also possible to use other DNA dyes (e.g. Hoechst 33258) orconventional methods such as the Tunel assay (Tdt-mediated XdUTP nickend labeling; DNA breaks), the detection of apoptotic enzymes (e.g.PARP, caspases) or proteins (e.g. p53, CD95), the translocation ofphosphatidylserine with fluorescent annexin or the detection of the DNAladder in an agarose gel.

It is possible with the disclosed method to find substances havinganti-apoptotic activity. These are preferably compounds which bindeither to receptors on the cell surface, particularly preferably IAPand/or the integrin α_(v)β₃ or to humoral factors, particularlypreferably TSP-1, in such a way that the specific interaction describedherein between TSP-1 and IAP and α_(v)β₃, which leads to induction ofapoptosis, does not take place, so that apoptosis cannot be induced andthus does not occur.

In a further preferred embodiment, these substances bind to IAP and thusprevent the apoptosis-specific calcium influx into the cell, so thatapoptosis cannot be induced and thus does not occur.

These inhibitors are suitable for producing pharmaceutical preparationswhich can be used in wound treatment for suppressing apoptosis offibroblasts, epithelial cells and other cells (e.g. smooth musclecells).

Active substances to be tested with the method of the invention are, inparticular, monoclonal antibodies and peptides which can easily beobtained with conventional methods of molecular biology and geneticmanipulation. However, it is clear that other substances having acorresponding effect can also be found using the test system of theinvention. The term “antibody” is used herein to describe both completeantibodies (i.e. antibodies having two heavy and two light chains) andfragments of antibodies having at least one antigen-binding site. Theidentification of an anti-TSP-1 antibody which has anti-apoptoticactivity on fibro-blasts is described in example 6. Many otherantibodies or antibody fragments can be tested in a similar way in orderto prevent the induction of apoptosis.

It is clear that it is possible to produce a whole series of peptidesand antibodies (antibody fragments) which can be identified as havinganti-apoptotic activity using the method of the invention in a similarway. In addition, it is also possible to identify other substances whichare not antibodies (antibody fragments) or peptides as havinganti-apoptotic activity with the aid of the method of the invention.

For the purposes of the present invention, the term “peptide” means asubstance which consists of one or more chains of a plurality, i.e. 2 ormore, amino acids connected by peptide linkages.

For the purposes of the present invention, the term “protein” means asubstance in which a plurality of “peptides” are connected together bypeptide linkages. This definition encompasses equally native proteinsand proteins which are at least partly “artificial”, and such“artificial” proteins can be modified for example by attachment ofchemical radicals which do not normally occur in native proteins to theamino acid chain.

For the purposes of the present invention, a peptide library means acollection of different peptides which can be investigated withoutfurther technical effort by the skilled worker for particular, specificbinding properties. Particularly well known to the skilled worker andtechnically extremely simple to manipulate are, for example, peptidelibraries based on the so-called phage display technology, in which upto 10⁷ and more phage particles each of which specifically express aparticular peptide on their surface may be present in a few millilitersof test liquid. If such phage particles are subjected to enrichment byaffinity chromatography, a biological factory for production, namely amonoclonal phage particle, i.e. an expression system, is also suppliedat the same time as the specifically binding peptides. Such systems areextremely well known to the skilled worker.

However, native peptides often show a low metabolic stability inrelation to peptidases and a relatively poor bioavailability.

Starting from the peptides shown above, the skilled worker is ablewithout inventive effort to develop a whole series of derived compoundswhich have a similar or identical mode of action and which are alsocalled inter alia peptidomimetics.

Compounds referred to as peptidomimetics for the purposes of the presentinvention are those which mimic the structure of peptides and are ableas ligands to imitate (agonist) or to block (antagonist) the biologicalactivity at the receptor/enzyme level. Peptido-mimetics ought inparticular to have an improved bioavailability and have improvedmetabolic stability. The nature of the mimesis may range from a slightlymodified initial structure up to a pure nonpeptide. See, for example, A.Adang et al., Recl. Trav. Chim. Pays-Bas 113 (1994), 63-78.

The possibilities available in principle for mimesis/derivatization of apeptide structure are as follows:

-   -   use of D- in place of L-amino acids    -   modification of the side chain of amino acids    -   modification/extension of the main peptide chain    -   cyclization to stabilize the conformation    -   use of templates which impose a particular secondary structure

Whereas the proteolytic stability of a peptide can be increased byreplacing L- by D-amino acids, modification of the side chains of one ofthe amino acids often leads to an improvement in the binding propertiesof the complete peptide.

When the peptide backbond is modified there is usually replacement of anamide group by amide-like groups (J. Gante, Angew. Chem. 106 (1994),1780-1802). It is possible by these measures to influence both thebinding affinity and the metabolic stability of the native peptide.

Cyclization of a linear peptide fixes its flexibility and thus itsglobal conformation. On fixation of the biologically activeconformation, the affinity of the peptide for the receptor is increasedbecause the decrease in entropion binding is less than on binding of aflexible linear peptide. For this purpose, amino acid side chains notinvolved in receptor recognition are linked together or to the peptidefragment.

The secondary structure of the peptide plays a crucial part in themolecular recognition of the receptor. Besides α-helix and β-pleatedsheet, important conformational elements are so-called turns as turningpoints in the peptide chain. Replacement of these structural units by aunit which stabilizes a definite secondary structure after insertioninto a peptide has led to the concept of the secondary structuremimetic.

It is also possible to increase the solubility of the peptides in waterfor example by introducing S- and C-glycopeptide derivatives. Furthermeasures may be, for example, PEGylation of the peptides.

The lipophilicity of hexapeptides can also be increased by, for example,attaching phenylalanines to the peptide sequence.

The cyclization and the N-terminal modification of peptides is describedfor example by Borchard, Journal of controlled Release 62 (1999),231-238 and by Blackwell et al., J. Org. Chem. 10 (2001), 5291-302 .

It is therefore clear that the skilled worker is able, starting from theknowledge provided by the present invention, easily to obtain a wholeseries of derived peptidomimetics, all of which are also encompassed bythe present invention, however.

In a further preferred aspect, the present invention therefore providespeptidomimetics which are derived from SEQ ID Nos 1-11, and substanceswhich comprise such peptidomimetics.

Particularly preferred substances usable according to the invention arelow molecular weight compounds. Such compounds often have only slight orno side effects if they are employed as active principle in apharmaceutical composition. A further advantage of such substances isthe possibility of oral administration.

Examples thereof are cyclic pentapeptides as described by Haubner etal., J. Am. Chem. Soc. 1996, 118, 7641-7472. Low molecular weightsubstances include according to the invention small peptides, aminoacids and amino acid analogs, steroids, nucleotides and other organicchemical substances having a molecular weight of ≦5 000, preferably ≦3000 and particularly preferably ≦2 000. [Haubner, R., Gratias, R.,Diefenbach, B., Goodman, S. L., Jonczyk, A., Kessier, II., Structuraland Functional Aspects of RGD-Containing Cyclic Pentapeptides as HighlyPotent and Selective Integrin α _(v)β₃ Antagonists, 118, 7461-7462(1996)].

Combinatorial libraries are likewise well known to the skilled worker. Alarge number of such libraries comprising a very large number of verydifferent molecules exist. It is possible to obtain from these librariesin automated processes low molecular weight compounds which bind veryspecifically, and it is clear that it is easily possible, in the lightof the invention presented here, to generate corresponding identificatesusable according to the invention.

Corresponding products are produced by conventional methods. Forexample, it is possible to dissolve peptides or antibodies (antibodyfragments), which are active ingredients of a pharmaceuticalpreparation, in a pharmaceutically acceptable carrier. One example of apharmaceutically acceptable carrier may be buffer solutions such asphosphate buffers or citrate buffers. It is also possible to maintainthe activity of the peptides by adding reagents which arepharmaceutically acceptable and, for example, maintain a reducingenvironment in the pharmaceutical preparation.

The specific dosage and posology for each patient depends on a number offactors, including the activity of the specific compounds used, the ageof the patient, the bodyweight, the general state of health, the sex,the diet, the time of administration, the route of administration, therate of excretion, the combination with other medicaments and theseverity of the individual disorder for which the therapy is applied. Itwill be established by a physician as a function of these factors.

Polypeptide medicaments, e.g. protein medicaments or antibodymedicaments, are normally administered parenterally, e.g. by aninhalation spray, rectally, by subcutaneous, intravenous, intramuscular,intra-articular and intrathecal injection and infusion techniques, orexternally in pharmaceutical formulations which comprise conventionalpharmaceutically acceptable carriers, adjuvants and vehicles. Otherroutes of administration are also suitable depending on the nature ofthe identified substance, e.g. orally.

The invention likewise provides pharmaceutical compositions whichcomprise an effective amount of a substance having anti-apoptoticactivity in combination with a conventional pharmaceutical carrier. Apharmaceutical carrier is, for example, a solid or liquid filler, anencapsulating material or a solvent. Examples of materials which can beused as pharmaceutical carriers are sugars such as lactose, glucose andsucrose; starch such as corn starch and potato starch; cellulose andderivatives thereof such as sodium carboxymethylcellulose,ethylcellulose and cellulose acetate; powdered tragacanth; malt;gelatin; tallow; medicament carriers such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil, sunfloweroil, sesame oil, olive oil, corn oil and soybean oil; polyols such aspropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol;esters such as ethyl oleate and ethyl laureate; agar; buffering agentssuch as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution, ethyl alcoholand phosphate buffer solutions, as well as other non-toxic compatiblesubstances which are used in pharmaceutical formulations. Washingagents, emulsifiers and glidants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, glidants, coating agentsand perfuming agents and preservatives may likewise be present in thepreparations according to the requirements of the pharmaceuticaltechnologist. The amount of the active ingredient combined with thecarrier materials in order to produce a single dose will vary dependingon the treated patient and the specific method of administration. Oneexample of such a pharmaceutical formulation is shown in example 19.

Production of a so-called living skin is described with great accuracyfor example in the applications EP 1 005 873 and WO 99/43787 of AdvancedTissue Sciences Inc., and the skilled worker is familiar with it. It ispossible to use for the purposes of the present invention artificialskin of whatever type if autologous or heterologous skin cells whichexpress CD47 or α_(v)β₃ on their surface are cultivated thereon, and ifthe matrix carrying the skin is stretchable.

EXAMPLES

The following examples explain the invention in more detail. However,they are not intended to be understood as restrictive.

ABBREVIATIONS

Abbreviations IAP: integrin-associated protein (CD 47) CBD: C terminalcell-binding domain of TSP-1 TSP-1: thrombospondin 1 RGD: Arg-Gly-AspHUVEC: human umbilical vein endothelial cells HPEC: human placentalendothelial cells DAPI: 4′,6′-diamidino-2-phenylindole MRC-5 fibroblastcell line Milli-Q purified water TNF-α tumor necrosis factor alpha bFGFbasic fibroblast growth factor

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: stretching apparatus, top view

FIG. 2: stretching apparatus, side view

FIG. 3: diagrammatic mode of functioning of the stretching apparatus(Ex. 16)

FIG. 4: determination of the effect of extension and compression of thecells on the apoptosis index (Ex. 16)

EXAMPLE 1 Cultivation of MRC-5 Fibroblasts (ATCC No.: CCL-171)

A) Reagents and Antibodies Used:

The RGD peptides, the CBD peptide and the Tsp1 are used as indicated inthe following table:

Incubation Biological Ligand Concentration time activity Humanthrombospondin 1  1 μg/ml (2−)24-72 h binds Prof. Dr. Vischer, integrinUniversität Münster, α_(v)β₃ and Institut für IAPArtherioskleroseforschung RGD cyclic 250 μg/ml (2−)24-72 h binds BachemBiochemica integrin GmbH, Heidelberg, α_(v)β₃ Germany Catalog No. H25740IAP peptide (CBD) 400 μg/ml (2−)24-72 h binds IAP Bachem BiochemicaGmbH, Heidelberg, Germany Catalog No. H1418

Specific Antibodies:

-   -   1.) Mouse monoclonal antibody against human CD47, from Cymbus        Biotechnology Ltd, UK, Catalog No.: CBL 489, 50 μg/ml.    -   2.) Monoclonal and polyclonal antibody against Tsp1, from Prof.        Dr. Vischer, Universität Münster, Institut für        Artherioskleroseforschung, Germany, 50 μg/ml.    -   3.) Monoclonal antibody against the integrin α_(v) subunit,        Chemicon International Inc., Canada, Catalog No.: MAB1960, 50        μg/ml.        B) Static, Dynamic and Tensioned Cultivation of MRC-5        Fibroblasts

Culture Medium for MRC-5:

-   -   89 ml of IF basal medium    -   10 ml of FCS (fetal calf serum)    -   1 ml of L-glutamine stock solution

IF Basal Medium

-   -   The IF basal medium is a 1:1 mixture of IMDM (Iscove's modified        Dulbecco's medium) and Ham's F12 medium.

L-glutamine Stock Solution

-   -   200 mM L-glutamine are dissolved in IF basal medium and        sterilized by filtration.        B1) Static Cultivation (Without Flow Stresses in the Cell        Culture Medium):

The MRC-5 cell line used is seeded in ungelatinized cell culturevessels. The subsequent cultivation takes place in an incubator at 37°C. and 5% by vol. CO₂ in a water vapor-saturated atmosphere. The culturemedium is changed every second to third day and, after confluence isreached, the cells are passaged with a division rate from 1:5 to 1:10.

B2) Dynamic Cultivation (Shear Stresses in the Cell Culture Medium):

The fibroblasts are initially cultivated under static conditions untilconfluence is reached. This is followed by cultivation under dynamicconditions in the cone and plate shear apparatus (after Bussolori et al.(1982) Rev. Sci. Instrum. 53, 1851-1854) for 24 h.

This is done by precultivation of the cells initially in gelatinizedculture dishes (◯=35 mm) with 0.15 ml/cm² culture medium and thenfurther cultivation under turbulent or laminar flow conditions in thecone and plate shear apparatus for a maximum of 24 h. The flowconditions result from the angle of the cone. The flow over the cells isturbulent at a cone angle of 5° and is laminar at a cone angle of 0.5°.

Solutions:

-   -   70% (v/v) ethanol    -   Culture medium for MRC-5 fibroblasts (see above)

Material:

-   -   Cone and plate shear apparatus (after Bussolori et al. (1982)        Rev. Sci. Instrum. 53, 1851-1854)

The shear stress generated is calculated from:

$\tau = \frac{{\mu \cdot 2}\;{\pi \cdot U_{display}}}{\alpha}$

-   τ=shear stress [dyn/cm]-   μ=viscosity of the medium at 37° C.-   α=cone angle-   U_(display)=speed of rotation displayed on the control element [rpm]

Procedure:

Before use, the cone and plate shear apparatus is cleaned with a softcloth and 70% (v/v) ethanol, the cone is sterilized and the apparatus isequilibrated at 37° C. in a heating cabinet. The precultivated cells arewashed with basal medium and provided with 0.1 ml/cm² fresh culturemedium, and the culture dish is rapidly fitted into the apparatus.

The cone is raised using the coarse adjustment, and the culture dishtogether with lid is inserted into the holder provided therefor. The lidis removed and the cone is adjusted over the culture dish. The exactdistance of the plate cone tip from the cell lawn is adjusted using amicrometer screw.

The scale of the micrometer screw shows in this case a value of 175. Atthis value, which was previously found empirically, the cone tip rotatesat a minimal distance from the cell lawn without abrading the latter.The assembled cone and plate shear apparatus is placed in the incubatorat 37° C., 5% (v/v) CO₂ and water vapor-saturated atmosphere for thelaminar or turbulent cultivation.

MRC-5 conditioned medium is produced as shown in Example 2.

B3) Cultivation in the Stretching Apparatus (Own Construction, FIGS. 1and 2):

The cells are precultivated initially on gelatinized silicone sheets (1)and, two days after confluence is reached, cultivated further in thestretching apparatus for the desired period.

Solutions:

-   -   Culture medium for MRC-5 fibroblasts (see above)

Material:

-   -   Stretching apparatus, cell culture dishes (diameter: 147 mm)        hexagon key, straight edge with millimeter scale, silcone sheet        of types LP 500-1, LP 500-3, LP 500-5; manufactured by        Laboratoire Perouse Implant, Bornel (France), marketed by        Aromando Medizintechnik GmbH, Düsseldorf, Germany

Procedure:

The stretching apparatus is initially cleaned with a soft cloth and 70%(v/v) ethanol and sterilized by autoclaving at 121° C. and 2 bar for 20min in a saturated water vapor atmosphere.

The sterile stretching apparatus is then placed in a cell culture dish(◯=147 mm) in such a way that the screws (2) which serve to fix thesilicone sheet (1) point upward. The silicone sheet is introduced usingforceps into the holder (3, 4) provided therefor and is fixed therein bytightening the screws of the holder (3, 4). The apparatus is turnedthrough 180° round its horizontal axis.

The sheet is tensioned by the required length through turning the pitchscrew (5), and the change in length is followed with the aid of astraight edge placed underneath the culture dish. Finally, 60 ml ofculture medium are put in the culture dish. The assembled stretchingapparatus is placed in the incubator at 37° C., 5% (v/v) CO₂ and watervapor-saturated atmosphere to cultivate the cells.

Cultivation in the Pulsatile Stretching Apparatus (Own Construction, seeFIG. 11):

The cells are stretched/compressed with a frequency of 1 Hz in thepulsatile stretching apparatus. This is achieved by a cone, which isactuated by a processor-controlled electric motor, actuating thefastenings of the silicone sheet as shown in FIG. 1 or 2 with anappropriate speed. The cells are initially precultivated on gelatinizedsilicone sheets and, two days after confluence is reached, cultivatedfurther in the pulsatile stretching apparatus for the desired period.

Solutions:

-   -   Culture medium for MRC-5 fibroblasts (see above)

Material:

-   -   Pulsatile stretching apparatus, cell culture dishes (diameter:        93 mm) hexagon key, straight edge with millimeter scale

Procedure:

The pulsatile stretching apparatus is initially cleaned with a softcloth and 70% (v/v) ethanol. The component into which the silicone sheetis clamped is sterilized by autoclaving at 121° C., 2 bar for 20 min ina water vapor-saturated atmosphere and placed in a cell culture dish(◯=93 mm) so that the screws which serve to fix the silicone sheet pointupward. The silicone sheet is introduced by means of forceps into theholder provided therefor and fixed in the latter by tightening thescrews of the holder. The apparatus is rotated by 180° around itshorizontal axis, and 15 ml of culture medium are added.

The cell culture dish is then inserted into the holder providedtherefor. The lid is removed, the eccentric is adjusted, and the speedof 1 Hz is set on the drive motor. The assembled stretching apparatus isplaced in the incubator at 37° C., 5% (v/v) CO₂ and watervapor-saturated atmosphere to cultivate the cells.

EXAMPLE 2 Production of Conditioned Medium

Solutions:

Culture medium for MRC-5 fibroblasts (see above)

Materials:

MRC-5 confluent

Greiner tubes

Procedure:

IF basal medium from example 1 is put onto a confluent MRC-5 cell lawnand conditioned for 48 hours-72 hours. The conditioned medium is thencentrifuged at 1000 g_(av) for 5 minutes. The conditioned medium isfrozen at −20° C. until used. The conditioned IF basal medium isemployed for apoptosis investigations. For this purpose it can besupplemented anew with 2 mM glutamine.

EXAMPLE 3 Determination of the Rate of Apoptosis by DAPI Staining ofApoptotic Cells

DAPI belongs to the group of indole dyes and is a selective DNA dye. Thedye is excited at 340-360 nm, and the emission maximum is at 480 nm. Itis employed for apoptosis investigations [cf. Cohen et al., ImmunologyToday, 14, No. 3, 126-130, 1993)].

Morphological Evaluation:

Solutions:

PBS (Phosphate Buffered Saline):

-   -   140 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄ and 1.5 mM KH₂PO₄ are        dissolved in water, with a pH of 7.2-7.4 being set up. The        resulting solution is sterilized by autoclaving.

Formaldehyde Solution:

-   -   4% (v/v) formaldahyde in PBS

DAPI Solution:

-   -   30 nM DAPI (Molecular Probes, Leiden, Holland) in MeOH, stored        at 4 C.

Materials:

-   -   Petri dish (35 mm) with MRC-5 fibroblasts in culture

Procedure:

The culture supernatant from a Petri dish is aspirated off, and the celllawn is fixed with 1 ml of formaldehyde solution on ice for 15 minutes,washed twice with 2 ml of PBS, treated with 0.5 ml of DAPI solution for15 minutes, washed with PBS and evaluated under the fluorescencemicroscope. The UV filter set and a 20× or 40× objective are used. 500-1000 cells are selected at random, and the cells with apoptotic nucleiare counted.

The apoptosis index is calculated by the following formula:

${{Apoptosis}\mspace{14mu}{{index}\mspace{14mu}\lbrack\%\rbrack}} = {\frac{{number}\mspace{14mu}{of}\mspace{14mu}{apoptotic}\mspace{14mu}{cells}}{{total}\mspace{14mu}{number}\mspace{14mu}{of}\mspace{14mu}{cells}}*100}$

EXAMPLE 4 Measurement of the Induced Calcium Influx into the Cells byUsing the Intracellular Calcium Indicator Fluo-3 AM

Fluo-3 is a calcium indicator which forms a fluorescent complex afterbinding of Ca²⁺. The ester Fluo-3 AM is taken up by the cell throughdiffusion. The calcium indicator Fluo 3 is produced in the cell onlyafter hydrolysis of the ester. Extracellular dye ester therefore doesnot impair the measurement. The measurement is carried out with normalfluorescein filters. At an excitation wavelength of 488 nm (500 nm), theemission maximum is at 525 nm and is increased by a factor of 100 (200)through calcium binding. The measurement range is between 0.05 and 20 μMfree Ca²⁺ [Merritt, J. E. et al., Biochem. J. 269, 513-519 (1990)].

Solutions:

PBS (see above)

Culture medium for MRC-5 fibroblasts (see above)

-   -   6 mM Fluo-3 AM stock solution (50 μg in 10 μl dimethyl sulfoxide        (DMSO); Molecular Probes, Leiden)    -   Fluo-3 AM concentration used: 3 μM in serum-free medium

Materials:

-   -   Cells in culture    -   24-well cell culture plates (microtiter plates)

Procedure:

Confluent MRC-5 cell lawns in 24-well plates are washed three times withserum-free medium, incubated with preheated serum-free medium withFluo-3 AM under culture conditions for 30 minutes and washed threetimes, and the plate is measured with a fluorimeter. Firstly thesensitivity of the instrument is adjusted, and then fluorescence valuescorresponding to the instantaneous calcium level in the cell arerecorded every 1-10 seconds. Substances can be added to the cells whilethe measurement is taking place. If a substance has an effect on thecalcium level, this can be recognized from the increase or decrease inthe fluorescence values. Assessment of the calcium level is possiblewith this method. The calcium influx is an early sign of apoptosistaking place.

EXAMPLE 5 Effect of Conditioned Medium on the Apoptosis of Staticallyand Dynamically Cultivated MRC-5 Fibroblasts

Solutions and Specific Materials:

see example 1

Procedure:

The used culture medium from a confluent MRC-5 fibroblast culture isremoved, and the cell lawn is washed once with culture medium andcultivated under the stated conditions for 24 h. The results obtainedare listed in the following table.

Cultivation Culture medium Apoptosis % static fresh 2.8 ± 0.2 staticconditioned 5.1 ± 0.2 dynamic fresh 0.5 ± 0.1 dynamic conditioned 0.5 ±0.1 dynamic fresh + TSP-1 0.4 ± 0.1

EXAMPLE 6 Effect of Thrombospondin-1 and Anti-Tsp1 Antibodies in theCulture Medium

Solutions and Specific Materials:

see example 1

Procedure:

The used culture medium from a confluent MRC-5 fibroblast culture isremoved, and the cell lawn is washed once with culture medium andcultivated under the stated conditions for 24 h. The results obtainedare listed in the following table.

Culture medium Apoptosis [%] fresh 1.6 ± 0.2 fresh + Tsp1 5.6 ± 0.2conditioned 5.1 ± 0.2 fresh + polyclon. anti-Tsp1 0.6 ± 0.2conditioned + polyclon. anti-Tsp1 0.6 ± 0.2 conditioned + monoclon.anti-Tsp1 0.6 ± 0.2

EXAMPLE 7 Change in the Apoptosis of MRC-5 Fibroblasts After Addition ofSpecific Peptides to the Culture Medium

Solutions and Specific Materials:

see example 1

Procedure:

The used culture medium from a confluent MRC-5 fibroblast culture isremoved, and the cell lawn is washed once with culture medium andcultivated under the stated conditions for 24 h. The results obtainedare listed in the following table.

Culture medium Apoptosis [%] fresh 1.6 ± 0.2 conditioned 5.1 ± 0.2fresh + Tsp1 5.6 ± 0.2 fresh + CBD + RGD/cycl. 5.7 ± 0.3

EXAMPLE 8 Tsp1 Secretion Under Static and Dynamic Culture Conditions (24h)

Procedure:

The used culture medium from an MRC-5 fibroblast culture 1 to 3 dayspost-confluence is removed, and the cell lawn is washed once withculture medium and cultivated under the stated conditions for 24 h. Theresulting medium is removed and centrifuged at 1 000 g_(av) at roomtemperature for 5 min, and the supernatant is used to determine the Tsp1content. The number of cells in the particular culture is found. Theresults obtained are listed in the following table.

Culture condition Tsp1 [ng/1 × 10⁶ cells] static 53.5 ± 6.1 dynamic 10.9± 0.5

EXAMPLE 9 Tsp1 Secretion Under Static Conditions as a Function of theDuration of Cultivation

Procedure:

The used culture medium from a confluent culture is removed, and thecell lawn is washed once with culture medium and cultivated furtherunder static conditions. The resulting medium is removed and centrifugedat 1000 g_(av) at room temperature for 5 min, and the supernatant isused to determine the Tsp1 content. The number of cells in theparticular culture is found. The results obtained are listed in thefollowing table.

Days, post confluence Tsp1 [ng/1 × 10⁶ cells] 1  7.8 ± 3.0 2 19.1 ± 2.53 21.2 ± 3.7 4 39.7 ± 3.8 5  99.2 ± 19.7 6 179.6 ± 16.3 7 192.4 ± 18.0

EXAMPLE 10 Effect of Anti-receptor Antibodies

Solutions and specific materials:

see example 1

Procedure:

The used culture medium from a confluent culture is removed, and thecell lawn is washed once with culture medium and cultivated under thestated conditions for 24 h. The results obtained are listed in thefollowing table.

Culture medium Apoptosis [%] fresh 1.6 ± 0.2 conditioned 5.1 ± 0.2conditioned + monoclon. anti-α_(v) 0.5 ± 0.3 conditioned + monoclon.anti-IAP 0.3 ± 0.2

EXAMPLE 11 Generation of Mechanical Tension by Isometric CollagenContraction in Fibroblast Cultures

1. Routine Cultivation, Two-Dimensional:

Culture Medium:

-   -   Culture medium for MRC-5 fibroblasts (see example 1)

Procedure:

The MRC-5 cell line used is seeded in ungelatinized cell culturevessels. The subsequent cultivation takes place in an incubator at 37°C. and 5% by vol. CO₂ in a water vapor-saturated atmosphere. The culturemedium is changed every second to third day and, after confluence isreached, the cells are passaged with a division rate from 1:5 to 1:10.

2. Cultivation in Collagen Matrices, Three-Dimensional:

The fibroblasts are initially cultivated under static conditions in 75cm² cell culture bottles until confluence is reached. The cells areharvested using 0.05% (w/v) trypsin/0.02% (w/v) EDTA in PBS.

2A. Production of the Collagen Gel (3 mg/ml):

Materials:

-   -   Vitrogen “100” collagen, 4° C., from COHESION Technologies,        INC., Palo Alto, Calif., USA    -   10× concentrated PBS stock solution, pH =7.4, with 0.005 mg/ml        phenol red    -   0.1 M HCl    -   0.1 M NaOH

Procedure:

The total volume is 2 ml per mixture: 0.2 ml of 10×PBS and 0.2 ml of 0.1M NaOH are added to 1.6 ml of Vitrogen “100” and the pH is adjusted to7.4 with 0.1 M HCl or 0.1 M NaOH.

2B. Cell Seeding in the Produced Collagen Gels:

2×10⁵ cells are seeded in each collagen matrix. For this purpose, 100 μlof the cell suspension (the appropriate cell titer is adjusted after thetrypsinization in serum-free DMEM) are introduced into 1.5 ml reactionvessels pre-equilibrated at 37° C., 100 μl of the neutralized collagensolution are added, and the resulting solution is incubated at 37° C.for 4 min. The collagen concentration is 1.5 mg/ml. The cell/collagensuspension is seeded in 24-well cell culture plates or in 3.5 cm culturedishes. The matrix is polymerized at 37° C. for 60 min.

2C. Generation of Mechanical Tension:

Material:

-   -   DMEM basal medium (Dulbecco's modified Eagel medium    -   Ascorbic acid stock solution (100×)        -   5 mg/ml ascorbic acid are dissolved in DMEM basal medium and            sterilized by filtration    -   Culture medium (tension) (per 100 ml)        -   89 ml of DMEM basal medium        -   10 ml of FCS        -   1 ml of ascorbic acid stock solution    -   Culture medium DMEM (per 100 ml)        -   90 ml of DMEM basal medium        -   10 ml of FCS

Procedure:

The matrices are covered with a layer of culture medium (DMEM+10% FCS+50μg/ml ascorbic acid) and cultivated in an incubator at 37° C. and 8.5%by volume CO₂ in a water vapor-saturated air atmosphere for 24 h.

Addition of ascorbic acid leads to an “isometric” contraction of thecollagen matrix, thus generating the mechanical tension on the cells.

The medium is changed after 24 h. Further cultivation takes place withDMEM+10% FCS (see above).

Cultivation of the Cells Under Mechanical Tension:

The matrix remains adherent to the bottom of the culture vessel for theperiod stated in each case.

Cultivation of the Cells Under Relaxed Conditions:

The adherent matrix is cautiously detached with a spatula from thesubstrate and “floats” in the culture medium without contact with thesubstrate. The tension is abolished. The cultivation takes place for theperiod stated in each case.

EXAMPLE 12 Effect of Mechanical Tension on the Apoptosis of MRC-5Fibroblasts

Procedure:

The cells are cultivated as stated in example 11. Day “0” corresponds tothe time after 24 h cultivation with ascorbic acid-containing mediumbefore further cultivation under tensioned or relaxed conditions. Afterthe stated times, the cell/collagen matrix is fixed in 4% (w/v)paraformaldehyde in PBS, followed by DAPI staining. The results obtainedare listed in the following table.

Cultivation Cultivation time Apoptosis [%] precultivation day “zero”, 0h  1.3 ± 0.2 relaxed 24 h 10.2 ± 0.7 relaxed 48 h 11.5 ± 0.5 relaxed 72h 11.2 ± 0.6 tensioned 24 h  2.4 ± 0.2 tensioned 48 h  1.7 ± 0.2tensioned 72 h  1.8 ± 0.3

EXAMPLE 13 Effect of Thrombospondin-1, of Anti-Tsp1 and Anti-ReceptorAntibodies on the Tension-Dependent Apoptosis of MRC-5 Fibroblasts

Substances and Antibodies Used:

Substances:

-   1. Purified Tsp1, from Dr. Vischer, Münster, Germany, 20 μg/ml.-   2. Chondramide A, a cytostatic isolated from myxobacteria, from    Prof. H. Reichenbach, GBF, Brunswick, Germany, 1 μM.

Specific Antibodies:

-   1. Mouse monoclonal antibody against human CD47, from Cymbus    Biotechnology Ltd, UK, 50 μg/ml (see example 1)-   2. Monoclonal and polyclonal antibody against Tsp1, from Dr.    Vischer, Munster, Germany, 50 μg/ml.-   3. Monoclonal antibody against the integrin β3 subunit, Chemicon    International Inc., Canada, Catalog No.: MAB1957, 50 μg/ml.

Procedure:

The precultivation, including the generation of the mechanical tension,takes place as described in example 11. “0 h” corresponds to the timeafter 24 h cultivation with ascorbic acid-containing medium beforefurther cultivation under tension and relaxed conditions and addition ofthe substances mentioned. The further cultivation time is 24 h. Theresults obtained are listed in the following table.

Apoptosis Cultivation Culture medium Period [%] Precultivation DMEM/FCS/Day “zero”,  1.1 ± 0.2 ascorbic acid  0 h tensioned DMEM/FCS 24 h  1.0 ±0.2 relaxed DMEM/FCS 24 h  6.2 ± 0.2 relaxed DMEM/FCS + mAb 24 h  0.5 ±0.1 IAP relaxed DMEM/FCS + mAb 24 h  0.9 ± 0.2 β3 relaxed DMEM/FCS + mAb24 h  0.5 ± 0.1 Tsp1 relaxed DMEM/FCS + Tsp1 24 h 10.5 ± 0.5

EXAMPLE 14 The Effect of Chondramide A on the Cytoskeleton of MRC-5(Cultivation in Collagen Matrices)

Procedure:

The precultivation and tensioning with ascorbic acid-containing culturemedium takes place as described in example 11. The procedure with thechondramide-containing sample is as follows: the ascorbic-containingmedium is removed and incubated with 1 μM chondramide A in culturemedium for 1 h. This is followed by detachment of the matrix from thesubstrate and a change of the culture medium. The further cultivationtime of all mixtures is 8 h. The results obtained are listed in thefollowing table.

Cultivation Culture medium Period Apoptosis (%) tensioned DMEM/FCS 8 h0.9 ± 0.2 relaxed DMEM/FCS 8 h 2.7 ± 0.2 relaxed DMEM/FCS + chondramideA 8 h 1.0 ± 0.2

EXAMPLE 15 Immunochemical Detection of IAP and the Integrin β₃ Subuniton MRC-5 Fibroblasts Under Various Culture Conditions

Procedure:

The precultivation takes place as described in example 11 (routinecultivation, two-dimensional:). The further cultivation under tensionand relaxed conditions takes place for 72 to 96 h.

Immunostaining:

-   -   removal of the culture medium    -   wash cell/collagen matrix 1× with PBS (37° C.)    -   fix 2% (w/v) paraformaldehyde in PBS at 4° C. for 30 min

The following steps are carried out on a Heidolph Duomax plate shaker(level 2) at room temperature.

-   -   2× in PBS for 5 min each time    -   saturate in 0.5% (v/v) Tween 20+0.5% (w/v) BSA in PBS for 1 h    -   wash 2× in 0.5% (v/v) Tween 20 in PBS for 5 min each time    -   incubation for 3 h with monoclonal mouse anti-human CD47 (Cymbus        Biotechnology Ltd, UK, see example 1) or monoclonal mouse        anti-human β3 antibody (Chemicon International Inc., Canada,        Catalog No.: MAB1957), 1:100 in 0.5% (v/v) Tween 20 in PBS    -   wash 3× in 0.5% (v/v) Tween 20 in PBS for 5 min each time

The following steps are carried out with protection from light:

-   -   incubation for 2 h with Cy3™ conjugated F(ab)2 fragment from        rabbit anti-mouse IgG (Dianova, Hamburg, Germany), wash 1:200 in        0.5% (v/v) Tween 20 in PBS    -   wash 3× in 0.5% (v/v) Tween 20 in PBS for 5 min each time.    -   cover collagen matrix with layer of PBS    -   fluorescence microscopic evaluation (FIG. 40×)

Note: two types of negative control are carried out:

-   a) in place of the specific CD47 antibody, nonspecific mouse IgG is    used in the same concentration.-   b) the cell lawn is incubated only with Cy3™ (Dianova, Hamburg,    Germany) conjugated F(ab)₂ fragment.

All stained cells were positive for the integrin. IAP was detectableonly on the cells cultivated under relaxed conditions. The cellcultivated under tensioned conditions show no IAP expression.

EXAMPLE 16 Detection of the Effect of Extension and Compression of theCells on the Apoptosis Index

In order to be able to investigate the effect of mechanical forcesresulting from the extension of the cells, cells are seeded on siliconesheets. One day after confluence is reached, the sheet is fitted in thestretching apparatus and extended by 20%. The apoptosis index isdetermined after incubation under culture conditions for 24 and 48 h.For compression of the cells, they are seeded on a sheet which hasalready been pretensioned. This sheet is relaxed by 20% one day afterconfluence is reached. This is shown diagrammatically in FIG. 3. Onceagain, the apoptosis index is determined after 24 and 48 h. The resultsare depicted in FIG. 4.

It is evident from FIG. 4 that the mechanical tension induced [lacuna]the cells by tensioning and relaxing the sheets has a marked effect onthe apoptosis index. The apoptosis index is considerably lower both inthe tensioned and in the compressed cells than in the controls. Thecomparatively low value of the tensioned sheet is noteworthy.

This can be explained by the material of the sheet allowing a slightrelaxation over the cultivation period, resulting in a minimalcompression of the cells which leads to a slight reduction in theapoptosis index. There is no significant difference evident betweentensioning or compression for one day and two days.

EXAMPLE 17 Determination of the Effect of Extension of the Cells on theProliferation and Apoptosis Index

Measurement of the Proliferation Index:

The proliferation rate is used to quantify the number of proliferatingcells obtained in an experiment. The definition of this is as follows:

${{PR}\mspace{14mu}\lbrack\%\rbrack} = {\frac{Z_{prolif}}{Z} \cdot 100}$

-   -   where PR=proliferation rates [%]    -   Z_(prolif)=number of proliferating cells    -   Z=total number of cells.

To measure the proliferation rate, the computer program “ZellCount” isused to count, in each of three experiments carried out independently ofone another, a sufficient number of fields of view to reach a totalnumber of 1 000 cells. The adjustment of the background is chosen sothat both the living and the apoptotic cells are counted.

Detection of proliferating cells by BrdU labeling: Solutions: Proteins:PBS MAb BrdU 70% ethanol Monclonal mouse anti-BrdU IgG 3N HClConcentration of the crude BrdU/dC solution fractions: 15 mM BrdU + 15mM dC in 20-250 μg/ml Milli-Q, Working concentration: 0.25 μg/ml storeat −20° C. in PBT-BSA Working dilution: 1:1000 in Mouse IgG culturemedium Total protein content: 13.8 mg/ml PBT is sterilized by filtrationafter PBS + 0.5% (v/v) Tween 20 making up in PBT-BSA PBT-BSA Workingconcentration: 25 μg/ml PBT + 0.5% (w/v) BSA in PBT-BSA DAPI solutionBT-KAM 30 nM DAPI in methanol biotinylated rabbit anti-mouse IgG Totalprotein content: 1.0 mg/ml Working dilution: 1:3000 in PBT-BSA Cys3 ™-SPCy3 ™ conjugated streptavidin Concentration: 1.8 mg/ml Working conc.: 5μg/ml in PBT-BSA Evaluation: Nikon Filterblock G Excitation wavelength:553 nm. Emission wavelength: 575 mn.

Procedure:

The cell lawn is incubated with 0.2 ml/cm² of the BrdU/dC solutiondiluted 1:1000 in culture medium for 30 min. The incubation is followedby washing twice with PBS, fixing the cell lawn with 0.2 ml/cm² 70% EtOHat 4° C. for 30 min and washing three times with PBS.

The following steps are carried out with gentle agitation (Duomax 1030,level 2) at room temperature:

The DNA is denatured by adding 0.2 ml/cm² 3N HCl for 20 min, which isfollowed by washing five times with PBS. 0.2 ml/cm² of the MAb-BrdU isadded and agitated for 30 min. The labeling is followed by washing fivetimes with PBT, adding 0.2 ml/cm² BT-Kam, incubating for 3 hours andwashing a further five times with PBT for 3 minutes. Staining takesplace by adding 0.2 ml/cm² Cy3-SP for one hour and is stopped by washingfive times with PBT for 3 minutes each time. To determine theproliferation rate it is necessary to carry out a DAPI counterstain andto evaluate the specimens after covering the cell lawn with a layer ofPBS under the fluorescence microscope.

A constant increase in the apoptosis and proliferation index is evidentwith the statically cultivated fibroblasts (FIG. 8).

EXAMPLE 18 Investigation of the Expression of the β₃ Subunit of theIntegrin α_(v)β₃ and of IAP Under Tensioned and Relaxed CultureConditions

One day after the tensioning of the silicone sheets, the MRC-5 are fixedand stained by means of indirect immunofluorescence staining in order tobe able to characterize the expression of the β₃ subunit of the integrinα_(v)β₃ and of IAP. The results of the stainings show that expression ofIAP on fibroblasts stretched by 20% is weaker than in cells cultivatedon the untensioned silicone sheet. This result is consistent with thepreviously measured apoptosis and proliferation indices.

EXAMPLE 19 Use of Antibodies or Peptides as Active Substances inPharmaceutical Formulations

The identified compounds having anti-apoptotic activity could beemployed as active substances in pharmaceutical formulations for thetreatment of traumatic conditions.

For this purpose, antibodies are expediently employed for example in aconcentration of 3-5 mg per ml in the following formulation:

-   -   water for injections    -   Polysorbate 80    -   disodium hydrogen phosphate/sodium dihydrogen phosphate    -   sodium chloride

This formulation is administered (sprayed on) as spray solution.

EXAMPLE 20 Incubation of Fibroblasts with Peptides Having Anti-apoptoticActivity

The cells are cultivated under static culture conditions as in example1/variant 1B1. The cells are seeded in the appropriate culture vessels(e.g. 24-well plate/0.5 ml per well) and employed for the test 3 daysafter complete confluence is reached. The cells are provided with newmedium:

-   (a) fresh culture medium [basic rate of apoptosis]-   (b) fresh culture medium with 1 μg/ml TSP-1 [apoptosis-inducing    substance; control]-   (c) culture medium (b)+peptide of SEQ ID NO 1; 1 mM-   (d) culture medium (b)+peptide of SEQ ID NO 2; 1 mM-   (e) culture medium (b)+peptide of SEQ ID NO 3; 1 mM-   (f) culture medium (b)+peptide of SEQ ID NO 4; 1 mM-   (g) culture medium (b)+peptide of SEQ ID NO 7; 1 mM-   (h) culture medium (b)+peptide of SEQ ID NO 8; 1 mM-   (i) culture medium (b)+peptide of SEQ ID NO 9; 1 mM-   (j) culture medium (b)+peptide of SEQ ID NO 10; 1 mM-   (k) culture medium (b)+peptide of SEQ ID NO 11; 1 mM-   (l) culture medium (b)+peptide of SEQ ID NO 5; 1 mM-   (m) culture medium (b)+peptide of SEQ ID NO 6; 1 mM

After incubation under culture conditions (example 1) for 24 h, thecells are fixed, stained with DAPI and examined morphologically underthe fluorescence microscope. The apoptotic cells and the total number ofcells are determined, and the apoptosis index is calculated (percent ofapoptotic cells). The data from 3 independent experiments are indicatedin the following table, indicating the averages and the standarddeviation.

The following peptides are tested:

Apoptosis Inhibition SEQ ID NO Amino acid sequence index [%] index [%] Kcontrol 4.23 ± 0.23 (1) R-A-Y-V-V-M 1.21 ± 0.29 71.4 ± 6.8 (2)R-W-Y-V-V-M 2.00 ± 0.29 53.7 ± 6.8 (3) R-Y-Y-V-V-M 1.45 ± 0.04 65.3 ±0.9 (4) R-E-Y-V-V-M 0.87 ± 0.32 80.4 ± 7.0 (7) R-G-Y-V-V-M 2.00 ± 0.59 53.7 ± 13.9 (8) R-M-Y-V-V-M 2.46 ± 0.29 42.8 ± 6.9 (9) R-T-Y-V-V-M 3.19± 0.50  25.4 ± 11.8 (10)  R-N-Y-V-V-M 3.70 ± 1.11  13.5 ± 16.2 (11) R-D-Y-V-V-M 2.66 ± 0.71  38.9 ± 16.7 (5) K-R-A-Y-V-V-M-W-K-K 0.42 ± 0.2190.1 ± 4.9 (6) K-R-E-Y-V-V-M-W-K-K 0.24 ± 0.18 94.4 ± 4.2 K: no peptideemployed

The apooptosis-inhibiting effect of these peptides is clearly evident,making them suitable for the use according to the invention for thetreatment of wounds.

The inhibition index of the peptides employed is calculated as follows:

Inhibition index [%]=100−(measured apoptosis index* 100/controlapoptosis index)

1. A medicament for promoting wound healing, comprising a substance thatbind to at least one of integrin-associated protein (IAP), integrinα_(v)β₃, and thrombospondin 1, wherein said substance inhibits bindingbetween thrombospondin 1 and at least one of IAP and integrin α_(v)β₃,and inhibit apoptosis characterized by nuclear fragmentation of cells,wherein said substance is a peptide consisting of the amino acidsequence depicted in SEQ ID NO
 1. 2. The medicament of claim 1, whereinthe substance binds to at least one of IAP and integrin α_(v)β₃ onfibroblasts and/or to thrombospondin 1 in such a way that the bindingbetween thrombospondin 1 and the at least one of IAP and integrinα_(v)β₃ is inhibited and the rate of apoptosis of the cells is reducedby more than 10%.
 3. The medicament of claim 2, wherein the cells areepithelial cells.
 4. The medicament of claim 2, wherein the cells arekeratinocytes.
 5. The medicament of claim 1 prepared by carrying out anidentification method comprising steps (i) to (v) , as follows: (i)culturing cells which express both IAP and integrin α_(v)β₃, (ii)causing the cells to produce an apoptosis-inducing material, and/oradding a material or materials inducing apoptosis, (iii) adding asubstance, (iv) measuring the rate of apoptosis, and (v) selectingidentificate substances which cause a reduced rate of apoptosis, andthen mixing the identificates with a pharmaceutically acceptablecarrier.
 6. The medicament of claim 1, wherein the substance is apeptide selected from a peptide library by prescreening with affinitychromatography using at least one of thrombospondin 1, IAP, and integrinα_(v)β₃ as a target.
 7. The medicament of claim 1, wherein the substanceis a low molecular weight active substance with a molecular weight of≦5000.
 8. The medicament of claim 7, wherein the low molecular weightactive substance is selected from a combinatorial library byprescreening with affinity chromatography using at least one ofthrombospondin 1, IAP, and integrin α_(v)β₃ as a target.
 9. Themedicament of claim 5, wherein the cells employed in the identificationmethod are endothelial cells.
 10. The medicament of claim 5, wherein thecells employed in the identification method are smooth muscle cells orfibroblasts.
 11. The medicament of claim 5, wherein the cells employedin the identification method are genetically modified cells whichexpress both IAP and α_(v)β₃ on their surface.
 12. The medicament ofclaim 5, wherein the cells employed in the identification method arecultivated under conditions under which consistently directed laminarflows do not occur.
 13. The medicament of claim 5, whereinthrombospondin 1 or an analogous compound with identical bindingproperties is added to the cell culture in the identification method.14. The medicament of claim 5, wherein the induction of apoptosis in theidentification method is determined by measuring increased calciuminflux into the cell.
 15. The medicament of claim 5, wherein the rate ofapoptosis in the identification method is determined by a methodselected from the group consisting of DAPI staining, TUNEL assay, DNAladder, annexin staining and enzyme detection.
 16. The medicament ofclaim 5, wherein the identificates bind to a calcium channel in the cellmembrane in such a way that apoptosis-specific calcium influx into thecells is suppressed.
 17. The medicament of claim 5, wherein theidentificates inhibit apoptosis-specific calcium influx intofibroblasts.
 18. The medicament of claim 5, wherein the processcomprises admixing at least one different fibroblast growth factor tothe medicament in addition to the indentificates.
 19. The medicament ofclaim 1, further comprising at least one fibroblast growth factor. 20.The medicament of claim 19, wherein the fibroblast growth factor isbasic fibroblast growth factor.
 21. The medicament of claim 1, furthercomprising a pharmaceutical acceptable carrier.